Group VIA calcium-independent phospholipase A 2 (iPLA 2 ) is a member of the PLA 2 superfamily that exhibits calcium-independent activity in contrast to the other two major types, secreted phospholipase A 2 (sPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ), which both require calcium for their enzymatic activity. Adenosine triphosphate (ATP) has been reported to allosterically activate iPLA 2 , and this has now been verified with a lipidomics-based mixed-micelle assay, but its mechanism of action has been unknown. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to identify ATP interaction peptide regions located within the ankyrin repeat domain at which ATP interacts. Molecular dynamics simulations revealed the mechanism by which ATP binds to its site and the main residues that interact. Site-directed mutagenesis was used to verify the importance of these residues in the role of ATP in regulating iPLA 2 activity. Importantly, calcium was found to abolish the enhancing regulatory function of ATP and to promote the inhibitory activity by calmodulin. Given previous evidence that calcium does not bind directly to iPLA 2 , its effect appears to be indirect via association with ATP and/or calmodulin. Using HDX-MS, we found that calmodulin interacts with the N terminus peptide region of iPLA 2 consisting of residues 20 to 28. These two regulatory iPLA 2 sites open the road to the development of potential targets for therapeutic intervention.

中文翻译：

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ATP 和钙调蛋白结合锚蛋白结构域对钙非依赖性磷脂酶 A 2 的变构调节机制


VIA 组钙非依赖性磷脂酶 A 2 （iPLA 2 ） 是 PLA 2 超家族的一员，与其他两种主要类型分泌的磷脂酶 A 2 （sPLA 2 ） 和胞质磷脂酶 A 2 （cPLA 2） 相比，它表现出钙非依赖性活性，它们都需要钙来发挥酶活性。据报道，三磷酸腺苷 （ATP） 可变构激活 iPLA 2 ，现在已通过基于脂质组学的混合胶束测定得到验证，但其作用机制尚不清楚。氢/氘交换质谱 （HDX-MS） 用于鉴定位于 ATP 相互作用的锚蛋白重复结构域内的 ATP 相互作用肽区域。分子动力学模拟揭示了 ATP 与其位点结合的机制以及相互作用的主要残基。定点诱变用于验证这些残基在 ATP 调节 iPLA 2 活性中的作用中的重要性。重要的是，发现钙可以消除 ATP 的增强调节功能并促进钙调蛋白的抑制活性。鉴于先前的证据证明钙不直接与 iPLA 2 结合，其作用似乎是通过与 ATP 和/或钙调蛋白的结合间接的。使用 HDX-MS，我们发现钙调蛋白与由残基 20 至 28 组成的 iPLA 2 的 N 末端肽区相互作用。这两个调节性 iPLA 2 位点为开发治疗干预的潜在靶点开辟了道路。